The basic objective is to elucidate the molecular mechanisms involved in the regulation of expression of the proopiomelanocortin (POMC) gene in pituitary and hypothalamic tissues. Using a series of rat POMC-specific cDNAs and gene clones, we propose to further characterize the transcriptional regulation of the POMC gene by steroids, peptides, and catacholamines, both singlely and in combination. Some studies will be done in the intact animal; however, certain studies will be done in primary cultures of pituitary cells and in AtT20 tumor cells to preclude indirect effects on POMC gene expression. The exact sites in the POMC gene involved with regulation by specific hormones of gene expression will be mapped by deletion analysis and site directed mutagenesis. We have already constructed a rat POMC promoter/rat Prolactin coding region construct to aid in these studies. Using protein/DNA binding studies using the regions identified in the previous studies and a reinitiating nuclear transcription assay, we will begin to identify and characterize the transacting factors which mediate the peptide hormone, neurotransmitter, and steroid mediated modulation of POMC gene expression. In related studies, we will characterize the induction of the glucocorticoid receptor encoding gene after culturing of the intermediate lobe cells. In addition to understanding how expression of the glucocorticoid receptor gene is suppressed in the intact animal, these studies will allow us to develop a relatively homogeneous primary culture system in which we can study the glucocorticoid/CRF regulatory interactions. We will further characterize the regulation of the POMC gene system in the periarcuate region of the hypothalamus using several reproductive paradigms. Studies will be performed both by solution hybridization assays as well as in situ hybridization assays, coupled with several anatomical techniques to functionally subdivide the POMC neurons. We will further develop the use of POMC gene intervening sequence specific DNA probes in solution and in situ hybridization as an indirect method for analyzing transcriptional rates in small amounts of tissues and individual cells, respectively. These studies should yield a great deal of understanding of the complex interaction of various types of hormonal substances in the regulation of a specific gene in a complex but normal physiological situation.